22 August 2006
Plasma Membrane I
Dr. Cynthia Smas, Ph.D.

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Functions of Plasma Membrane

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• Eukaryotic cells are surrounded by a lipid bi-layer as are membrane-bound organelles
• Functions of the plasma membrane
  • Regulate nutrient and ion transport
  • Regulate transport of waste
  • Maintain correct chemical conditions in the cell
  • Provide a site for lipid-based chemical reactions
  • Interact with other cells and extracellular matrix (ECM)
  • Detect and transducer signals from environment to cell

Fluid Mosaic Model

• Phospholipid bilayer with imbedded proteins, carbohydrates, etc. serving the functions of the plasma membrane
• Membrane is fluid and asymmetrical
• Components:
  • Lipids
    • Form a permeability barrier
    • Define basic architecture
  • Protein
    • Define the unique functions of the membrane
    • Determine selective permeability
    • Functions as transporters, channels, junctions
    • Important for energy uptake and signal transduction

Lipids

• Lipids are water-insoluble biomolecules
• Highly soluble in organic solvents
• Variety of structures
• Used for fuel and energy storage
• Signaling functions
• Components of membranes
• Membrane Lipids have an amphipathic nature
  • Hydrophilic (polar) head group
  • Hydrophobic (non-polar) acyl side chains with fatty acid hydrocarbon (tails)
• Types of membrane lipids
  • Phospholipids
    • Composed of 4 groups
      • Hydrophobic Components
        • Fatty-Acid side chains
        • Glycerol
      • Hydrophilic Components
        • Phosphate
        • Alcohol
  • Commonly Occurring Membrane Phospholipids
    • Phosphatidyl serine
    • Phosphatidyl choline
    • Phosphatidyl ethanolamine
    • Phosphatidyl inositol
  • Glycolipids
    • Glucose or sugar unit attached to the glycerol group instead of a phosphate with alcohol
  • Cholesterol
    • Common in the plasma membrane of animals
    • Very small hydrophilic region with –OH group
    • Mostly hydrophobic
• Aqueous media phospholipids and glycolipids ready form a bilayer sheet
  • Two faces of plasma membrane (leaflets):
    • Exoplasmic – toward extracellular environment
    • Cytoplasmic – towards the intracellular environment
  • Energetically favorable and stable because of hydrophobic interactions by water, van der Waals attractive forces between hydrocarbon tails, and electrostatic and hydrogen bonding interactions between polar head groups and water molecules

Micelles and Liposomes

• Miscelles are spheres of lipids
• Solublization and purification of membrane proteins
• LDL and bile particles are mixed micelles
• Liposomes are hollow spheres of lipids with hydrophilic regions on the inner and outer surfaces
  • Used in functional study of membrane proteins
  • Important for drug delivery and therapeutics

Architecture of the Membrane

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• Semi-permeable membrane
  • Steroids, gases, ethanol, water and urea can pass through lipid membrane
  • The rest cannot pass because they are too large, too polar or charged, or both
• Membrane Proteins
  • Carry out most membrane processes

Peripheral Proteins

• Loosely associated with the membrane
• Removable with mild conditions (changes in salinity or pH)
• Do not enter or span the hydrophobic core of membrane

Integral Proteins

• More tightly associated by the membrane
• Removable only with harsh conditions (detergents)
• Spans or enter the hydrophobic core of the membrane
• Structures that allow integral proteins to enter or span the plasma membrane:
• β-barrel used in bacteria
• α-Helixes used in eukaryotes
  • Hydrophobic amino acid residuals (sequence of 20-25)
  • Hydrophobicity allows protein to span the lipid core
    • Example: Glyophorin uses an α-Helix to span the membrane
• Predicting transmembrane spanning regions of a protein
  • Plot hydrophobicity score of primary structure of protein
  • Regions rich in hydrophobic amino acids likely to be the membrane spanning region
  • Hydrophilic amino acids unlikely to be in transmembrane region, though it can happen
  • Proline not likely to be in transmembrane region because it disrupts the α-helix structure
    • Example: Prostaglandin H Synthase – intergral protein that enters but does not span the membrane
  • Proteins relying on specific lipid structures to associate with the plasma membrane
    • Attach a lipid anchor to protein to “dock” it to the membrane
• Variety of membrane protein interactions give strength and flexibility to the plasma membrane

Diseases Associated with Defective Plasma Membrane

• Defect in protein localization on plasma membrane
  • Leads to disease as proteins are not in the right place to act effectively
    • Example: GPI anchor in Acquired Hemolytic Anemia
      • Acquired because genetic disease occurred in stem cells that mutated and gave rise to progeny of defective cells
    • Example: Hereditary Spherocytosis
      • RBC functionality is closely tied to membrane integrity
      • Defects in RBC membrane proteins are often indicated by visible alterations in RBC morphology
      • Mutations in genes for Spectrin, Ankyrin, etc. leads to weakened interaction of peripheral and integral membrane proteins.
      • Cytoskeleton architecture altered – RBC’s more fragile
      • Spherocytic cells clog up the spleen where they are destroyed
• Cancer
  • Alterations to membrane protein and/or lipids key to metastases and invasion throughout the body
  • MDR membrane transporter protein is bases for developing multi-drug resistance during chemotherapy
• Diabetes
  • Defective insulin signaling and/or function of glucose transporters
• Heart Disease
  • Defective cell-cell communication

Studying Membrane Proteins

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• Reconstitution of membrane proteins in artificial liposome to study protein activity in natural environment
  • Difficult studies because of purification steps and danger of denaturing membrane protein
• Membrane Proteins as Drug Targets
  • Designing drugs to target protein “active sites”
  • Predict how mutations in membrane protein gene may impact function

Membrane Fluidity

• Influence arrangement of proteins and lipids
• Foster assembly/disassembly of protein subunits and signaling complexes in membrane
• Changes membrane permeability
• Excessive fluidity leads to membrane destruction
• Altering fluidity can alter membrane and/or cell function

Fatty Acid Composition

• Unsaturated hydrocarbon tails have kinks causing membranes to be more fluid
• Saturated hydrocarbon tails can be packed tightly and be more viscous
• Mixture of unsaturated and saturation used to produce optimally fluid membranes

Temperature

• Increased fluidity with increasing temperature
• Longer acyl chains or saturated change would make membranes more viscous
• Shorter acyl chain or desaturation would make membranes more fluid
• Longer the acyl chain, the higher the melting temperature

Cholesterol

• Key regulator of membrane fluidity in animals
• Disrupts regular interactions of fatty acyl side chains
• Reduces likelihood of undergoing a phase transition – high cholesterol abolishes phase transition
• Below melting temperature – cholesterol increases fluidity
  • Introduces kinked structure that disrupts packing
• Above melting temperature – cholesterol decreases fluidity
  • Limits overall free movement of the lipid side chains due to planar shape
• Lipid raft – cholesterol-rich microdomains of the plasma membrane

Mobility of Membrane

• Lateral diffusion – move left or right
• Transverse diffusion – very rare and energetically unfavorable
• Rotational diffusion – spin, essentially
• Proteins also have mobility
  • Depends on the size of molecule, interactions with other molecules, temperature, lipid composition, and protein composition

Fluorescence Recovery After Photobleaching

• Use antibody to fluorescently label protein
• Location of protein is indicated by fluorescent signal viewed under a fluorescent microscope
• Signal in a specific area can be “bleached” out by a laser
• Movement of protein in the bleached area can be monitored
  • As proteins move into the bleached area, the bleached area will “recover” fluorescent signal
  • Intensity of bleach and recovery can be determined

Membrane Asymmetry

• Two surfaces of membrane have different proteins associated
• Asymmetric distribution occurs for both lipids and proteins on both exoplasmic and cytoplasmic surfaces
  • Asymmetry is necessary for proper function
  • Choline-containing phospholipids are mostly exoplasmic
  • Amino-containing phospholipids are mostly cytoplasmic
  • Flippases, floppases, and scramblases can impact lipid asymmetry
  • Fatty acyl side chains also show leaflet enrichment
  • RBC – cytoplasmic leaflet enriched in unsaturated fatty acyl chains

Altered Distribution of Membrane Lipids

• Can target cells for destruction
• Exposure of phosphatidyl serine on the exoplasmic leaflet occurs in physiological and pathologic states
  • Examples: Platelet activation and aggregation, Recognition and removal of cells, Apoptosis (programmed cell death)

Protein Topology

• Membrane proteins have specific and consistent topology
  • Determined at the time of synthesis in the ER is maintained at the plasma membrane
  • Topology of membrane proteins is maintained and don’t “flip-flop”

Protein Glycosylation

• Generally, only exoplasmic portion of proteins are glycosylated
• Complex sugar groups added in lumen of ER and Golgi
  • Sugar can be linked to asparagine (N-link) or serine (O-link)
• Confers specificity and function
  • Example: Blood group antigens

Membrane Protein Repertoire

• Each cell type has a unique repertoire of membrane proteins
• SDS-PAGE Analysis – separate proteins based on size
  • Can determine different repertoires of membrane proteins from different sources

Objectives

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